Environments contaminated with the chlorinated solvents trichloroethylene (TCE) and dichloroethylene (DCE) are major cleanup problems, and will involve hundreds of millions in Superfund expenditures in the coming years. TCE is a common ground-water contaminant in the United States as a result of solvent spills and dry-cleaning chemical disposal. Cis-1,2-dichloroethylene (DCE) is also a common ground-water contaminant that originates from anaerobic dehalogenation of TCE in the environment. These compounds are potential carcinogens and cannot be removed effectively from ground water using conventional water purification processes. For many sites, bioremediation is the only practical approach for cleanup, but the use of previously known solvent-metabolizing microorganisms has often been hindered by their extreme sensitivity to the toxic effects of intermediates produced as the result of these bacteria's degradation of TCE and related compounds, and by their sensitivity to high concentrations of TCE itself.
Various types of bacteria have been shown to degrade TCE, including methanotrophs, toluene-degraders, phenol-degraders, propane oxidizers, and nitrifiers. (Fogel et al., 1986; Wackett et al., 1989; Ensley et al., 1991). These bacteria degrade TCE by a process of "cometabolic degradation," in which substrates that support growth induce nonspecific monooxygenase or dioxygenase enzymes that fortuitously can also degrade TCE and other chlorinated aliphatic compounds (CACs). The dioxygenases and monooxygenases both require oxygen and reducing power in the form of NADH.
When methanotrophs, the most extensively studied of this type of bacteria, metabolize trichloroethylene (TCE), dichloroethylene (DCE), or vinyl chloride by means of oxidative cometabolism, the resulting chemical intermediaries are known to be toxic to these microorganisms at relatively low levels (e.g., Alvarez-Cohen and McCarty, 1991a; Alvarez-Cohen and McCarty, 1991b; Alvarez-Cohen and McCarty, 1991c; Rasche et al., 1991; Oldenhuis et al., 1989). Moreover, the presence of methane competitively inhibits TCE degradation by methanotrophs (Strand et al., 1990). Consequently, the use of methanotrophs in bioremediation is severely limited.
Bacteria other than methanotrophs are also known to degrade CACs. For example, Burkholderia (Pseudomonas) cepacia G4 (U.S. Pat. Nos. 4,925,802, and 5,071,755) expresses elevated levels of a CAC-degrading toluene ortho-monooxygenase enzyme in the presence of phenol, toluene, o-cresol, or m-cresol (originally called "Pseudomonas cepacia G4", this bacterium has been reclassified as "Burkholderia cepacia G4," Yabuuchi et al., Microbiol. Immunol. 36:1251-1275, 1981). This bacterium, isolated from an industrial waste site water sample, is strictly aerobic. The G4 isolate is reportedly a gram-negative, rod-shaped bacterium that grows predominantly in pairs and short chains, and can utilize carbohydrates as a carbon source. This organism reportedly consumes TCE at a rate of about 2.5 nmol/minute per mg of protein with a K.sub.S of 3 .mu.M (Folsom et al., 1990). "Intermediate toxicity," i.e., toxicity resulting from CAC degradation products, was observed in studies of Pseudomonas putida F1, as evidenced by a decrease in growth rate (Wackett and Householder, 1989). This study also reported that TCE damaged the P. putida's intracellular proteins.
In other studies, phenol was utilized in situ to stimulate natural microorganisms to degrade CACs (Hopkins et al., 1993a; Hopkins et al., 1993b). Phenol-degrading microorganisms were readily stimulated, and phenol removal from the injection site was complete after 170 hours. Cometabolic degradation of TCE and DCE was also observed, and phenol-degrading microorganisms were found to remove TCE more effectively than did methanotrophic organisms stimulated by methane addition in concurrent tests. Some laboratory studies with phenol/toluene oxidizers have been done (Folsom et al., 1990; Nelson et al., 1988; Shields et al., 1989; Wackett and Householder, 1989), although such bacteria are less well-studied than the methanotrophs. However, it has been demonstrated that the toluene-degrader Pseudomanas putida F1 suffers toxic cellular effects from TCE degradation byproducts (Wackett and Householder, 1989). Another phenol-degrading bacteria reported to metabolize TCE is Alcaligenes eutrophus JMP134 (Harker and Kim, 1990).